This project begins a study of the biosynthesis of carnitine in the mammalian organism. Carnitine is known to derive from lysine either in free or peptidyl form. Trimethyllysine then forms and becomes hydroxylated in the Beta-position. The resultant hydroxy compound is cleaved oxidatively to gamma-butyrobetaine which is then hydroxylated by an alpha-ketoglutarate-dependent dioxygenase to form carnitine. We intend first to study the final hydroxylation leading to carnitine. We have developed an assay based on the detritiation of tritiated gamma-butyrobetaine. Using that assay, the purification of the hydroxylase from calf liver will be monitored. Ultimately the purified enzyme will be studied for kinetics and mechanism of action. Whether hydroxylation occurs stereospecifically with or without retention of configuration, whether a peroxy or other kind of derivative intervenes will also be studied. By use of substrate amounts of enzyme we shall attempt to uncouple the decarboxylation of alpha-ketoglutarate from the hydroxylation of gamma-butyrobetaine. We shall try to determine why catalase and other proteins enhance the hydroxylation. Then we shall study the hydroxylation of trimethyllysine, and, if not determined in the meanwhile by other investigators, the oxidative cleavage of hydroxytrimethyllysine to gamma-butyrobetaine. At all points we shall keep sight of the biomedical significance of these studies, namely that interference with any of the steps of biosynthesis of carnitine may cause abnormal metabolism of lipids in liver, kidney, skeletal muscle, heart and other tissues and organs. We shall study whether cells in culture accumulate carnitine, synthesize it, or in fact are deficient in it; and how any one of these circumstances then influences lipid metabolism of the cells.